Chemical and serological properties of lipopolysaccharides from Vibrio parahaemolyticus O-untypeable strains isolated from patients

Chemical and serological studies have been carried out on the O-antigenic lipopolysaccharides (LPS) of six strains, U-6443, W-90144, X-3972, AD-7999, 90A-6611 and KX-V212, of Vibrio parahaemolyticus isolated from patients. The O-serotypes of these strains have not been identified because they were not agglutinated by any diagnostic antisera against known O-serotype strains. A compositional sugar analysis of their LPS revealed that out of the six O-untypeable (OUT) strains, U-6443, W-90144 and AD-7999 strains belonged to chemotype II (chemotype of O2), 90A-6611 and KX-V212 strains to chemotype III (chemotype of O3, O5, O11 and O13) and X-3972 strain to chemotype IV (chemotype of O4). A structural analysis of LPS isolated from KX-V212 revealed that the inner core region of the LPS consisted of only one mole of 2-keto-3-deoxy-D-manno-octonic acid, which carried a phosphate group at position C4 and the outer core at position C5. In passive hemolysis tests performed by using LPS as the antigen to sensitize sheep red blood cells (SRBC), and diagnostic antisera (O1 to O11) or anti-whole-cell rabbit antisera raised against O12, O13 and the six OUT strains, strong cross-reactivity was observed among LPS derived from the strains belonging to chemotype II (U-6443, W-90144, AD-7999 and O2). Strong cross-reactivity was also observed between X-3972 (chemotype IV) and O4 LPS. In contrast, LPS from two of the strains belonging to chemotype III (90A-6611 and KX-V212) did not react with any of the antisera raised against known O-serotypes. Cross-absorption tests showed that the O-antigens of U-6443, W-90144 and AD-7999 were identical to that of O2, and the O-antigen of X-3972 to that of O4. On the other hand, after the absorption of antisera raised against 90A-6611 and KX-V212 with O2 cells, the hemolytic activities against SRBC sensitized with homologous LPS were still retained at a high titer, whereas the hemolytic activities against SRBC sensitized with LPS from other O-serotype strains were completely eliminated. A cross-absorption test revealed that the O-antigens of these two strains were identical to each other. Thus, it was demonstrated that the O-serotype of OUT strains 90A-6611 and KX-V212 was not involved in the known O-serotypes; rather it represented a novel serotype which has not hitherto been reported.     

Structural diversity and endotoxic activity of the lipopolysaccharide of Yersinia pestis

The endotoxic activity of the lipopolysaccharides (LPS) with defined chemical structure from Yersinia pestis strains of various subspecies differing in their epidemic potential was studied. The LPS of two strains of Y. pestis ssp. caucasica and ssp. altaica, whose structures have not been studied earlier, were analyzed by high-resolution mass spectrometry. In addition to reported structural changes, an increase in the degree of LPS phosphorylation was observed when strain I-2377 (ssp. altaica) was cultivated at an elevated temperature. A high tumor necrosis factor alpha(TNF-alpha)-inducing activity observed for LPS samples from Y. pestis cultures grown at 25 degrees C correlated with an increased degree of lipid A acylation, particularly, with the presence of the hexaacyl form of lipid A, which was absent from the LPS when bacteria were cultivated at 37 degrees C. No correlation was found between the lethal toxicity of the LPS in vivo and its ability to induce TNF-alpha production in vitro.     

Genetic diversity of Bradyrhizobium strains isolated from Arachis hypogaea

Rhizobia are used exclusively in agricultural systems for enhancing the ability of legumes to fix atmospheric nitrogen. Knowledge about the indigenous population is necessary for the selection and application of inoculant strains. In this study, we have assessed the genetic diversity of Bradyrhizobium strains isolated from the host plant, Arachis hypogaea along the coastline of Tamil Nadu. Different populations collected from varying environmental conditions were analysed for salt and pH tolerance. Genetic diversity among the strains was studied using RAPD markers and PCR-RFLP of 16S rDNA and nifD genes. The approaches used in this study yielded consistent results, which revealed a high degree of heterogeneity among strains and detection of two distinct genetic groups.     

Structural analysis of the lipid A isolated from Hafnia alvei 32 and PCM 1192 lipopolysaccharides

Hafnia alvei, a Gram-negative bacterium, is an opportunistic pathogen associated with mixed hospital infections, bacteremia, septicemia, and respiratory diseases. The majority of clinical symptoms of diseases caused by this bacterium have a lipopolysaccharide (LPS, endotoxin)-related origin. The lipid A structure affects the biological activity of endotoxins predominantly. Thus, the structure of H. alvei lipid A was analyzed for the first time. The major form, asymmetrically hexa-acylated lipid A built of β-d-GlcpN4P-(1→6)-α-d-GlcpN1P substituted with (R)-14:0(3-OH) at N-2 and O-3, 14:0(3-(R)-O-12:0) at N-2′, and 14:0(3-(R)-O-14:0) at O-3′, was identified by ESI-MSⁿ and MALDI-time-of-flight (TOF) MS. Comparative analysis performed by MS suggested that LPSs of H. alvei 32, PCM 1192, PCM 1206, and PCM 1207 share the identified structure of lipid A. LPSs of H. alvei are yet another example of enterobacterial endotoxins having the Escherichia coli-type structure of lipid A. The presence of hepta-acylated forms of H. alvei lipid A resulted from the addition of palmitate (16:0) substituting 14:0(3-OH) at N-2 of the α-GlcpN residue. All the studied strains of H. alvei have an ability to modify their lipid A structure by palmitoylation.     

Iron requirement and siderophore production in Bradyrhizobium strains isolated from Acacia mangium

Fourteen Bradyrhizobium strains isolated from nitrogen-fixing Acacia mangium trees which originated from different geographical areas were analysed for their iron requirement and siderophore production. All strains were affected by starvation but responded differently to it. The increase in bacterial cell yield in response to iron supplementation, as well as the strain's sensitivity towards the synthetic iron-chelator 2,2-dipyridyl, suggested a discrimination of these strains into two groups. Four strains in one group, including a reference strain, produced siderophore(s) when grown under starvation. Other strains belonging to the second group were characterized by a lower iron requirement, a higher sensitivity to 2,2-dipyridyl, and did not apparently demonstrate any siderophore production. Two of the siderophore-producer strains, as well as a Rhizobium reference strain, excreted citrate, which was under iron regulation. Citrate was shown to facilitate iron incorporation in strains of either group.     

AFLP fingerprint analysis of Bradyrhizobium strains isolated from Faidherbia albida and Aeschynomene species

The diversity of Bradyrhizobium isolates from Faidherbia albida and Aeschynomenee species was assessed using AFLP analysis, a high-resolution genomic fingerprinting technique. Reference strains from Bradyrhizobium japonicum, Bradyrhizobium elkanii and Bradyrhizobium liaoningense were included for comparison. At a similarity level of 50%, a total of 34 different groups were obtained by cluster analysis of the genomic fingerprints. Four of these clusters correspond to the three reference species, demonstrating the large diversity of the isolates studied. Comparison with other data demonstrates that AFLP has a higher resolution than restriction analysis of 16S rRNA genes, SDS-PAGE analysis of proteins and phenotypic analysis. Results of the latter two methods showed little correspondence with the genotypic data.     

Isolation and characterization of the lipopolysaccharides from Bradyrhizobium japonicum.

The lipopolysaccharide (LPS) of Bradyrhizobium japonicum 61A123 was isolated and partially characterized. Phenol-water extraction of strain 61A123 yielded LPS exclusively in the phenol phase. The water phase contained low-molecular-weight glucans and extracellular or capsular polysaccharides. The LPSs from B. japonicum 61A76, 61A135, and 61A101C were also extracted exclusively into the phenol phase. The LPSs from strain USDA 110 and its Nod- mutant HS123 were found in both the phenol and water phases. The LPS from strain 61A123 was further characterized by polyacrylamide gel electrophoresis, composition analysis, and 1H and 13C nuclear magnetic resonance spectroscopy. Analysis of the LPS by polyacrylamide gel electrophoresis showed that it was present in both high- and low-molecular-weight forms (LPS I and LPS II, respectively). Composition analysis was also performed on the isolated lipid A and polysaccharide portions of the LPS, which were purified by mild acid hydrolysis and gel filtration chromatography. The major components of the polysaccharide portion were fucose, fucosamine, glucose, and mannose. The intact LPS had small amounts of 2-keto-3-deoxyoctulosonic acid. Other minor components were quinovosamine, glucosamine, 4-O-methylmannose, heptose, and 2,3-diamino-2,3-dideoxyhexose. The lipid A portion of the LPS contained 2,3-diamino-2,3-dideoxyhexose as the only sugar component. The major fatty acids were beta-hydroxymyristic, lauric, and oleic acids. A long-chain fatty acid, 27-hydroxyoctacosanoic acid, was also present in this lipid A. Separation and analysis of LPS I and LPS II indicated that glucose, mannose, 4-O-methylmannose, and small amounts of 2,2-diamino-2,3-dideozyhexose and heptose were components of the core region of the LPS, whereas fucose, fucosmine, mannose, and small amounts of quinovosamine and glucosamine were components of the LPS O-chain region.     

Changes in electrophoretic profiles of lipopolysaccharides from competitive strains of Bradyrhizobium spp. induced by soybean roots

The soybean Bradyrhizobium strain Semia 566 was introduced into soils of the Cerrados (Brazilian edaphic savannas) in the late 1960s. Then, nodule occupancy by this strain was not greater than 2%. Recently, this serogroup has been found in approximately 60% of nodules formed on soybeans cultivated in the Cerrados, replacing the strains 29W and Semia 587, the Brazilian commercial inoculant for soybean. Although some re-isolates of Semia 566, adapted to Cerrado soils, were more competitive than 29W under both field and aseptic conditions, they did not differ from the parental strain, based on their lipopolysaccharide (LPS) electrophoretic profile. The only exceptions were the isolates 4A-5 and CPAC-15 which presented an additional polysaccharidic band of low molecular weight or higher mobility. On the other hand, this same band may be induced and intensified in LPS extracted from competitive strains (29W, 220, 204, 370, 372, 516, 122 and CPAC-15) after bacterial contact with soybean roots for 6 or 12 h. In addition, a 29W Tn5 mutant with a phenotype of delayed nodulation showed a delayed induction of this polysaccharidic band. Conversely, the LPS of less competitive strains was not modified or showed a weak intensification of this band. As this band alteration was correlated with the concurrent elevation of dominance in nodules, it may be suggested that LPS plays a role in the competitive ability of rhizobia strains for nodulation.     

Chemical composition of lipopolysaccharide from Azospirillum lipoferum

Abstract The lipopolysaccharide isolated from Azospirillum lipoferum strain SpBr17 (ATCC29709) was proven to be composed from 7 neutral sugars, two of which, rhamnose and glucose, were the major constituents. Two heptoses, l -glycero- d -mannoheptose and d -glycero- l -mannoheptose were identified. Among 8 fatty acids isolated from the lipopolysaccharide only 3-hydroxypalmitic acid was amide-bound. The approximate molar ratios of the constituents 3-deoxy- l -mannooctulosonic acid : glucosamine : amide-linked fatty acids : ester-linked fatty acids : phosphate were 0.8 : 4 : 2 : 4 : 2.5.     

Immunochemical and partial chemical characterization of fractions of membrane-bound smooth lipopolysaccharide-protein complex from Brucella abortus

Smooth lipopolysaccharide (sLPS) of Brucella abortus was prepared and fractionated by a modification of the procedures of Moreno et al. (J. Bac. 138:361–369, 1979). Washed B. abortus cells were disrupted by 21 freeze-quick thaw cycles with ultrasonication to separate the non-membrane-bound material. Ultrasonicated bacteria were used for preparation of membrane-bound sLPS ( f5, the main crude sLPS fraction described by Moreno et al.). Phenol extraction was repeated 3 times and then washed with H₂O 10 times to remove most of the chromogen, polysaccharides and nucleic acids, eliminating the need for enzyme treatment as described previously. The membrane-bound sLPS was fractionated into 3 to 5 groups according to the extent of dialysis and centrifugation, these fractions required only 80 ng for positive ELISA, about 0.2 ng for positive Limulus lysate tests, and reacted well with precipitating antibodies in the serum from a strain 2308 infected cow. They had marked differences in precipitin curves and chemical composition. The protein content varied from 16% to 42% as determined by dye binding test and 17 to 60% by Lowry phenol method using bovine serum albumin as the standard, which implies that the proteins associated with LPS may also play important roles in the complex for the immunochemical interactions and the heterogeneity of B. abortus lipopolysaccharide protein complex. As compared with previous reports, a higher yield of sLPS, ranging from 3.6% to 7.7% of dried bacteria, was obtained. Group f5A, which had a standard bell shaped curve in the precipitin assay, is one of the major fractions in all three strains (1119.3, 19 and 2308). The amount of other subfractions obtained varied with batches or strains of B. abortus. These results provide a new profile of the immunochemical reactivities and the heterogeneity on B. abortus smooth membrane-bound endotoxins.Abbreviations LPS lipopolysaccharides - sLPS smooth lipopolysaccharides - cLPS crude lipopolysaccharides - AH acid hapten - KDO 3-deoxyoctulosonic acid - ELISA enzyme-linked immunosorbent assay - f5 the main crude sLPS described by Mooreno et al. [2] - LAL limulus amoebocyte lysate test - HexN Hexosamine - PS phenol sulfuric method - O orcinol method     

Occurrence and taxonomic significance of oxo-fatty acids in lipopolysaccharides from members of Mesorhizobium

Lipopolysaccharides (LPSs) isolated from seven strains of Mesorhizobium were studied for the presence of fatty acids with particular attention for 27-oxooctacosanoic acid and 4-oxo fatty acids. The LPSs from all analysed strains contained various amounts of 27-oxo-28:0 and all of them, with the exception of Mesorhizobium tianshanense, contained also 4-oxo fatty acids (4-oxo-20:0, 4-oxo-i-21:0, 4-oxo-22:0). The group of amide-linked fatty acids consisted of a wide range of 3-hydroxylated and 4-oxo fatty acids whereas all the nonpolar as well as the (omega-1) hydroxylated long-chain acids and the 27-oxo-28:0 fatty acids were ester-linked. The characteristic spectrum of 3-hydroxy fatty acids and presence of 27-OH-28:0 as well as 27-oxo-28:0 acid in LPSs of Mesorhizobium showed that these strains were closely related. Therefore the lipid A fatty acid pattern could be a useful chemotaxonomic marker which helps to isolate the Mesorhizobium group from rhizobium bacteria during the classification process.     

A rapid, small-scale procedure for the structural characterization of lipid A applied to Citrobacter and Bordetella strains: discovery of a new structural element

Endotoxins [lipopolysaccharides (LPSs)] are part of the outer cell membrane of Gram-negative bacteria. Their biological activities are associated mainly with the lipid component (lipid A) and even more specifically with discrete aspects of their fine structure. The need for a rapid and small-scale analysis of lipid A motivated us to develop a procedure that combines direct isolation of lipids A from bacterial cells with sequential release of their ester-linked fatty acids by a mild alkali treatment followed by MALDI-MS analysis. This method avoids the multiple-step LPS extraction procedure and lipid A isolation. The whole process can be performed in a working day and applied to lyophilized bacterial samples as small as 1 mg. We illustrate the method by applying it to the analysis of lipids A of three species of Citrobacter that were found to be identical. On the other hand, when applied to two batches of Bordetella bronchiseptica strain 4650, it highlighted the presence, in one of them, of hitherto unreported hexosamine residues substituting the lipid A phosphate groups, possibly a new camouflage opportunity to escape a host defense system.     

Endotoxic activity of lipopolysaccharides isolated from emergent potential cystic fibrosis pathogens

Improved antimicrobial therapies against the classical spectrum of pathogenic bacteria which colonise the lungs of cystic fibrosis (CF) patients has resulted in improved life expectancy and quality of life. Bacterial species that are resistant to a broad range of antibiotics including Stenotrophomonas maltophilia and Alcaligenes xylosoxidans have now emerged as potential new pathogens to fill the niche. At present, it is unclear from clinical data whether these microbes are commensal or pathogenic. In this study we have quantified the inflammatory potential of lipopolysaccharide (LPS) from eight species of Gram-negative organisms which have been cultured with increasing frequency from CF patients. Inflammatory responses induced by LPS from whole human blood and a human-derived monocyte cell line (THP-1) were assessed. Enzyme-linked immunosorbent assays were used to detect interleukin-6, interleukin-8, and tumour necrosis factor alpha (TNF). A bioassay was also used to assess TNF activity. With the exception of S. maltophilia, LPS extracted from all of the bacteria tested upregulated, by varying degrees, expression of each of the proinflammatory cytokines assayed. This study represents the first comprehensive report of the endotoxic potential of a new wave of microbes which are associated with CF.     

Biological activity of lipopolysaccharides from clinical Bacteroides fragilis strains isolated in Poland determined in reaction with limulus amoebocyte lysate

The aim of this study was to determine a biological activity of lipopolysaccharides (LPS) from clinical Bacterioides fragilis strains isolated in Poland by means of quantitative, photometric BET (LAL) method with Limulus polyphemus amoebocyte lysate and chromogenic substrate S-2423. Lipopolysaccharides were extracted from nine clinical B. fragilis strains by the procedure of Westphal and Jann (1965). Crude LPS preparations were purified with ultracentrifugation. Biological activities of bacterial endotoxins were determined by quantitative BET method with chromogenic substrate S-2423 (ENDOCHROME kit). Tests were performed according to the recommendations of the producer (Charles River Endosafe Ltd., USA). E. coli O55:B5 LPS and LPS preparations from reference B. fragilis strains were applied to compare the results of examinations. Activities of endotoxins from clinical B. fragilis strains isolated in Poland determined in reaction with Limulus amoebocyte lysate were differentiated. Among endotoxins of clinical B. fragilis strains the most active was the preparation from strain cultured in the case of pancreatic ulcer (B. fragilis 80/81 LPS). Lipopolysaccharides of examined B. fragilis strains were less active in BET test than E. coli O55:B5 LPS.     

Symbiotic effectiveness of Bradyrhizobium strains isolated from Parasponia and tropical legumes on Parasponia host species

The symbiotic effectiveness of Bradyrhizobium strains isolated from three species of Parasponia and from legumes were compared on Parasponia grown in Leonard-jars. Effectiveness of each symbiotic association was estimated from dry weight and total nitrogen of shoots and nodules of plants grown on medium free of combined nitrogen. Twenty strains isolated from three species of Parasponia were found to vary in their effectiveness on P. andersonii, the least effective fixing one fifth of the nitrogen of the most effective strains. The outcome of the symbiosis was not associated with the host source of the test strain. P. andersonii, P. rugosa and P. rigida responded differently to a selection of seven strains of Parasponia Bradyrhizobium; some strains were either ineffective or fully effective on each host, while others varied in their symbiotic performance. P. andersonii fixed significantly (P < 0.001) larger quantities of nitrogen than either P. rugosa or P. rigida with p. rigida being the least effective. In contrast to Bradyrhizobium strains from Parasponia spp. which formed nodules rapidly (within 11–20 days), nine strains isolated from legumes required between 25 and 74 days to form partially effective nodules. The thre Parasponia species formed relatively large quantities of nodule tissue relative to the amount of nitrogen fixed and shoot dry matter produced. The Bradyrhizobium isolated from Parasponia plants growing in Papua New Guinea soils could be grouped together on the basis of their infection characteristics on Parasponia and legumes.     

Inhibitory effect of galectin-3 on the cytokine-inducing activity of periodontopathic Aggregatibacter actinomycetemcomitans endotoxin in splenocytes derived from mice

Galectins, a family of animal lectins, are involved not only in development and differentiation but also in immunoregulation and host–pathogen interactions. Galectin-3 interacts with lipopolysaccharides in gram-negative bacteria such as Escherichia coli, Salmonella minnesota and Pseudomonas aeruginosa . The present study investigated whether galectin-3 inhibited the cytokine-inducing activity of periodontopathic bacterial lipopolysaccharides using splenocytes derived from mice of different ages. Lipopolysaccharides were extracted from Aggregatibacter actinomycetemcomitans Y4 and Porphyromonas gingivalis ATCC 33277, and then purified. Enzyme-linked immunosorbent assay (ELISA) analysis revealed that galectin-3 adhered to A. actinomycetemcomitans lipopolysaccharides, but not to the lipopolysaccharides of P. gingivalis . Splenocytes were prepared from 1- or 7-month-old C57BL/6 mice. Either A. actinomycetemcomitans lipopolysaccharides (200 ng mL⁻¹) alone or lipopolysaccharides and murine galectin-3 (10 μg mL⁻¹) were added to culture solutions, and the release of interleukin-6 (IL-6) and interferon-γ (IFNγ) from splenocytes was measured by ELISA after a 17-h incubation. In all mice tested, A. actinomycetemcomitans lipopolysaccharide stimulation significantly increased the production of IL-6 and IFNγ ( P <0.01). Murine galectin-3 suppressed lipopolysaccharide-induced cytokine production in the splenocytes of the 1-month-old mice ( P <0.02 for IL-6; P <0.05 for IFNγ), but not in the splenocytes of the 7-month-old mice. This suggests that responses change with age.     

Two rhizobial strains, Mesorhizobium loti MAFF303099 and Bradyrhizobium japonicum USDA110, encode haloalkane dehalogenases with novel structures and substrate specificities

Haloalkane dehalogenases are key enzymes for the degradation of halogenated aliphatic pollutants. Two rhizobial strains, Mesorhizobium loti MAFF303099 and Bradyrhizobium japonicum USDA110, have open reading frames (ORFs), mlr5434 and blr1087, respectively, that encode putative haloalkane dehalogenase homologues. The crude extracts of Escherichia coli strains expressing mlr5434 and blr1087 showed the ability to dehalogenate 18 halogenated compounds, indicating that these ORFs indeed encode haloalkane dehalogenases. Therefore, these ORFs were referred to as dmlA (dehalogenase from Mesorhizobium loti) and dbjA (dehalogenase from Bradyrhizobium japonicum), respectively. The principal component analysis of the substrate specificities of various haloalkane dehalogenases clearly showed that DbjA and DmlA constitute a novel substrate specificity class with extraordinarily high activity towards beta-methylated compounds. Comparison of the circular dichroism spectra of DbjA and other dehalogenases strongly suggested that DbjA contains more alpha-helices than the other dehalogenases. The dehalogenase activity of resting cells and Northern blot analyses both revealed that the dmlA and dbjA genes were expressed under normal culture conditions in MAFF303099 and USDA110 strain cells, respectively.     

Linear bactenecin analogs with cell selectivity and anti‐endotoxic activity

Bactenecin (Bac) is a 12‐residue disulfide‐linked antimicrobial peptide isolated from the granules of bovine neutrophils. In this study, to develop novel linear Bac analogs with cell selectivity and anti‐endotoxic activity, we designed and synthesized a series of linear Bac analogs with amino acid substitution in Cys^3,11 and/or Val^6,7 of Bac. Among Bac analogs, some analogs (Bac‐W, Bac‐KW, Bac‐L, Bac‐KL, Bac‐LW, and Bac‐KLW) with higher hydrophobicity showed the amalgamated property of cell selectivity and anti‐endotoxic activity. Furthermore, Bac‐W, Bac‐KW, Bac‐LW, and Bac‐KLW showed serum stability comparable with that of disulfide‐bonded Bac. Therefore, these Bac analogs (Bac‐W, Bac‐KW, Bac‐LW, and Bac‐KLW) can serve as promising antibiotics for the development of therapeutic agents for treatment against endotoxic shock and bacterial infection. In addition, our results suggest that a little increase in hydrophobicity may be responsible for the decreased cell selectivity of the multiple Arg‐containing peptides (Bac‐W, Bac‐L, and Bac‐LW) over the multiple Lys‐containing peptides (Bac‐KW, Bac‐KL, and Bac‐KLW). Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.